Vitamin D (Vit D) insufficiency and deficiency is a significant health issue. Our lab has been conducting pioneering studies examining the role of Vit D in the eye, with a focus on the cornea. Exciting emerging data point to the therapeutic potential of Vit D in treating ocular pathologies. The general hypothesis is that Vit D deficiency or insufficiency can lead to exacerbated anterior segment-related ophthalmic problems brought on by a primary insult or disease. Specific Aim 1. Examine the influence of Vit D deficiency on corneal epithelial wound healing in diabetic mice. Rationale. Diabetes is a significant complicating factor in any number of pathological conditions, and diabetic keratopathy effects approximately 70% of all diabetics. The hypothesis for Aim 1, based on novel preliminary data collected using diabetic VDR knockout mice, is that Vit D deficiency will lead to a significantly exacerbated deficit in the corneal wound healing of diabetic mice. Approach. Cornea epithelial wounds will be made in control and vitamin D receptor (VDR) knockout diabetic mice. Healing rates will be compared and corneas will be examined for morphological differences. The influence of Vit D metabolites on wound healing in these VDR knockout diabetic mice will be determined. Specific Aim 2. Examine effects of 24,25D on corneal epithelial cell function. Rationale. We previously demonstrated that the concentration of 24,25D is the highest of all Vit D metabolites in the eye. Recent studies indicate that 24,25D is not just an inactive metabolite. Our preliminary studies demonstrate that 24,25D may play a significant role in the function of the corneal epithelium. Approach. The influence of 24,25D on corneal epithelial cell proliferation and migration will be studied using cell culture. 24,25D effects on the expression of VDR and the key Vit D metabolic-related enzymes CYP27B and CYP24A1 will be measured using qPCR and western blotting. 24,25D signaling through VDR will be examined using VDR knockout mice and cell culture. Integrated with Aims 1 and 3, respectively, we will examine the influence of 24,25D on corneal epithelial wound healing along with gap junction function and desmosomes. Specific Aim 3. Determine how Vit D influences corneal epithelial intercellular junctions. Rationale. We previously demonstrated that Vit D influences the function of corneal epithelial gap junctions and tight junctions. We will test the hypothesis, based on our preliminary studies, that the gap junction functional effects we reported are directly related to modified density and distribution of specific gap junction proteins and that the weak epithelial attachment we observe in VDR knockout mice is related to changes in desmosome protein density and distribution. Approach. Determine the influence of VDR knockout on corneal epithelial mRNA levels, density, and distribution of gap junction- and desmosome-specific proteins. We will also examine the influence of VDR knockout on corneal limbal cell gap junction diffusion coefficients using the FRAP method we recently described. We will also determine if Vit D supplementation affects gap junction connectivity using cell culture scrape loading and FRAP measurements.